The response to injury is an innate host immune response for restoration of tissue integrity. Wound healing, whether initiated by trauma, surgery, microbes or foreign materials, proceeds via an overlapping pattern of events including coagulation, inflammation, epithelialization, formation of granulation tissue, matrix and tissue remodeling. The process of repair is mediated in large part by interacting molecular signals, including cytokines that motivate and orchestrate the manifold cellular activities which underscore inflammation and healing.
The initial injury triggers coagulation and an acute local inflammatory response followed by mesenchymal cell recruitment, proliferation and matrix synthesis. Failure to resolve the inflammation can lead to chronic non-healing wounds, whereas uncontrolled matrix accumulation, often involving aberrant cytokine pathways, leads to excess scarring and fibrotic sequelae.
Most types of injury damage blood vessels, and coagulation is a response to initiate hemostasis and protect the host from excessive blood loss. Vessel wall injury exposes collagen to elements of flowing blood. Collagen is a thrombogenic surface component and has been shown to be a stimulant for platelet adhesion, aggregation and the release of their granules leading to the recruitment of (Ruggeri, Z. M. et al.; Seminars in Hematology, 1994, 31, 229-39) additional platelets to this area to form aggregates or a thrombus. The initial contact of the platelets to the vessel surface is mediated by collagen bound von Willebrand Factor (vWF) and a specific vWF receptors on platelets, the glycoprotein Ib-V-IX complex. This reversible adhesion allows platelets to roll over the damaged area, which is then followed by a firm adhesion mediated by the collagen receptors (alpha(2)beta(1), GPVI) in addition resulting in platelet activation. This leads to the conformational activation of the platelet alpha(IIb)beta3 receptor, fibrinogen binding and finally to platelet aggregation. In addition ADP, epinephrine and circulating clotting factors drive the further activation process of platelets while simultaneously an increase in thrombin activity contributes to the formation of the cross-linked fibrin clot. Platelet-platelet aggregation supports this process and is driven by fibrinogen as a mediator that bridges cells through the glycoprotein IIb/IIIa receptor.
This normal physiological response plays a role in the course of pathological processes where platelets adhere to collagen exposed in sclerotic lesions (Van der Rest M. et al.; FASEB Journal, 1991, 5, 2814-23) and start to build-up occlusions. Depending on the location and extent of the occlusion complications such as myocardial infarction, stroke, inflammation or pulmonary embolism may be the outcome of this process.
As a direct acting antithrombotic agent heparin which blocks the thrombin activity, thus preventing the formation of fibrin rich thrombi, is currently used in anti-thrombotic interventions. Heparin is used in indications such as: unstable angina and acute myocardial infarction. However, several short comings of heparin such as intravenous application, requirement for anti-thrombin-III as a cofactor, reduced affinity for clot-bound thrombin, it's inactivation by several plasma proteins, the occasional induction of thrombocytopenia and it's biological heterogeneity remain unresolved.
Recent development of low molecular weight heparin has contributed a version for subcutaneous application; however the therapeutic benefit over the standard heparin has been modest. Unfortunately, the same applies to the other directly acting antithrombins such as hirudin, hirulog and warfarin. One of the problems seems to be related to the increased production of thrombin under antithrombotic treatment (Rao, A. K et al., Circulation, 1996, 94, 389-2395).
Other recent strategies have therefore been focused to the process of prothrombin activation which is driven by Factor Xa. The challenge is the design of appropriate inhibitors directed to this factor.
Another panel of therapeutics is represented by the thrombolytic regimens and has been focused on the development of staphylokinase, streptokinase, urokinase type plasminogen activator, tissue type plasminogen activator and anisoylated-plasminogen-streptokinase activator complex. The differences in time necessary to inducing reperfusion is different for each of these thrombolytic agents, however the contribution in terms of reducing the overall mortality is equal for all the products. In addition, reocclusion and/or prolonged bleeding are some of the complications. This might be due to relatively low specificity for fibrin and the short plasma half-life of these compounds.
A complication arises when artificial surfaces come in contact with blood. When this is the case, there is increased tendency to induce thrombotic events by activation of platelets and/or induction of coagulation. These effects may cause failure of vascular grafts, cardiac valves, stents, catheters or any other blood contacting device or material. The protein disclosed herein has the ability to create non-thrombogenic surfaces and can therefore be further exploited by immobilization of this protein to the materials and devices described above. Such a treatment renders such materials or devices biocompatible and thromboresistant.
The two lines of therapy, which are currently being used in an attempt to control platelet adhesion, activation and subsequent thrombosis and intimal hyperplasia, are anti-platelet agents and anti-thrombotic administration. Although drugs such as aspirin effectively block the synthesis of Thromboxane A2 through inhibition of the cyclooxygenase pathway, they do not prevent the collagen-induced platelet adhesion and activation, which stimulate the development of intimal hyperplasia. The use of heparin as an antithrombotic agent is associated with complications and limitations including a non-predictable dose response, need for close laboratory monitoring, limited activity against clot bound thrombin, multiple inhibitory sites, antithrombin III dependency, a risk of bleeding, as well as a need for continuous infusion. Clearly, an ideal therapeutic agent would be one that produces site specific and localized effects without systemic distribution or a generalized coagulopathy.
Due to the limitations associated with the available antithrombotic agents there is an actual need for new alternative strategies and therapeutics. Therefore, the need for new and improved therapeutics and methods for inhibiting the events in the pathophysiology of platelet adhesion is obvious, and contributions in this field are expected to decrease morbidity and mortality associated with angioplasitic or surgical procedures.